Abstract

The epigenetic silencing of the O6-methylguanine-DNA-methyltransferase (MGMT) gene by promoter hypermethylation is emerging as a clinically relevant predictor of response to treatment in glioma patients. MGMT promoter hypermethylation can be detected in approximately half of gliomas and it is associated with longer overall survival in patients who receive alkylating chemotherapy in association with radiotherapy. MGMT methylation status was determined by real time Quantitative Methylation Specific PCR (QMSP) in 50 glioma samples (28 formalin fixed paraffin-embedded and 22 snap frozen specimens). The specificity of the assay was evaluated by analyzing normal brain tissues from 28 healthy individuals. Results from QMSP analysis were compared with data obtained on the same samples by MSP. QMSP analysis demonstrated a statistically significant difference in both methylation level (P=0.000009 Mann Whitney Test) and frequencies (P=0.0000007, Z-test) in tumour samples as compared with normal brain tissues. Based on ROC curve analysis an optimal cut off value of 0.35 was used to distinguish between methylated and unmethylated samples. Of the 50 glioma samples 29 showed methylation levels above the cut off value resulting in a sensitivity of 58% (95%CI: 45%-71%). Only two normal brain tissues showed methylation levels above the cut off value resulting in a specificity of 93% (95%CI: 84%-100%). No significant differences in the performance of the test were detected between paraffin embedded samples and snap frozen specimen. Conventional MSP analysis showed similar sensitivity than QMSP (56%) but much lower specificity (64%). Our results indicate that QMSP analysis is a highly specific method for MGMT promoter methylation detection in glioma samples and suggest that it may represent a powerful tool to identify glioma patients that will benefit from alkylating agents chemotherapy.